The pipeline for drugs for control and elimination of neglected tropical diseases: 1. Anti-infective drugs for regulatory registration

The World Health Organization ‘Ending the neglect to attain the Sustainable Development Goals: A road map for neglected tropical diseases 2021–2030’ outlines the targets for control and elimination of neglected tropical diseases (NTDs). New drugs are needed to achieve some of them. We are providing an overview of the pipeline for new anti-infective drugs for regulatory registration and steps to effective use for NTD control and elimination. Considering drugs approved for an NTD by at least one stringent regulatory authority: fexinidazole, included in WHO guidelines for Trypanosoma brucei gambiense African trypanosomiasis, is in development for Chagas disease. Moxidectin, registered in 2018 for treatment of individuals ≥ 12 years old with onchocerciasis, is undergoing studies to extend the indication to 4–11-year-old children and obtain additional data to inform WHO and endemic countries' decisions on moxidectin inclusion in guidelines and policies. Moxidectin is also being evaluated for other NTDs. Considering drugs in at least Phase 2 clinical development, a submission is being prepared for registration of acoziborole as an oral treatment for first and second stage T.b. gambiense African trypanosomiasis. Bedaquiline, registered for tuberculosis, is being evaluated for multibacillary leprosy. Phase 2 studies of emodepside and flubentylosin in O. volvulus-infected individuals are ongoing; studies for Trichuris trichuria and hookworm are planned. A trial of fosravuconazole in Madurella mycetomatis-infected patients is ongoing. JNJ-64281802 is undergoing Phase 2 trials for reducing dengue viral load. Studies are ongoing or planned to evaluate oxantel pamoate for onchocerciasis and soil-transmitted helminths, including Trichuris, and oxfendazole for onchocerciasis, Fasciola hepatica, Taenia solium cysticercosis, Echinococcus granulosus and soil-transmitted helminths, including Trichuris. Additional steps from first registration to effective use for NTD control and elimination include country registrations, possibly additional studies to inform WHO guidelines and country policies, and implementation research to address barriers to effective use of new drugs. Relative to the number of people suffering from NTDs, the pipeline is small. Close collaboration and exchange of experience among all stakeholders developing drugs for NTDs may increase the probability that the current pipeline will translate into new drugs effectively implemented in affected countries. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-022-05581-4.


Preventive chemotherapy
Depending on (1) co-endemicity of loiasis and onchocerciasis due to risk of adverse reactions to ivermectin and diethylcarbamazine and (2) status of MDA programme. Countries without onchocerciasis or loiasis: · Annual diethylcarbamazine (6 mg/kg) with 400 mg albendazole (DA) · Annual Ivermectin, diethylcarbamazine plus albendazole (IDA) in areas with less than four two drug treatment rounds; not having met stopping criteria with DA despite having met coverage targets; with infections suggesting local transmission post MDA or post-validation. [25] Eligible population excludes pregnant women, children under 2 years of age, and the severely ill. [11] Countries with onchocerciasis (due to risks of ocular adverse reactions to diethylcarbamazine in O. volvulus infected individuals, for review (Awadzi et al., 2015)) in areas without loiasis co-endemicity · Annual ivermectin (150-200 ug/kg) with 400 mg albendazole (IA), except in areas where biannual ivermectin treatment is being delivered for onchocerciasis [25] Eligible population excludes pregnant women, children <90 cm (≈15 kg), severely ill individuals [11] Countries with onchocerciasis where loiasis is co-endemic (due to risks of severe adverse reactions to ivermectin in individuals with high Loa loa microfilaraemia, [26,27])

Macrofilaricide, drug safe in Loa loa infected individuals
Page 10 of 53

Preventive chemotherapy
Endemic communities with ≥10% Schistosoma spp infection prevalence: · Annual treatment with a single dose of all ≥ 2 years of age targeting ≥75% treatment coverage · Consideration should be given to twice yearly preventive chemotherapy in areas with demonstrated lack of appropriate response to annual preventive chemotherapy or high endemicity areas with baseline prevalence ≥50% in school-age children Endemic communities with <10% Schistosoma spp infection prevalence with ongoing preventive chemotherapy: · Continuation with same or reduced frequency towards interruption of transmission Test and treat approach In endemic communities with <10% Schistosoma spp infection Dose: 40 mg/kg, based on height pole for people ≥94 cm or ≥ 4 years Eligible population: all ≥2 years, including pregnant women after the first trimester and lactating women [11,29] Improved praziquantel and pediatric formulation. New drugs to complement praziquantel in case of resistance.
Eligible population: all young (12-23 months), preschool (24-59 months), schoolage children, non-pregnant adolescent girls and women of reproductive age, pregnant women in 2nd and 3rd trimester, lactating women [11,30] · WHO is preparing guidelines for addition of ivermectin where prevalence of S. stercoralis exceeds 10% and in areas with high prevalence of T. trichiura [1] Case management · Treatment of individuals living in areas endemic for STH and S. stercoralis [1] Selected complementary core interventions [1] · WASH in case of emergence of drug resistance Rabies · Rabies lyssavirus https://www.who.int/healthtopics/rabies None.
Case management of confirmed or suspected cases of human rabies · Thorough wound washing · Post-exposure prophylaxis (PEP) with the rabies vaccine immediately after exposure to a potentially rabid animal · Rabies immunoglobulin for category III exposures immediately after exposure to a potentially rabid animal · Palliative care Selected complementary core interventions [1] · Vaccination of dogs and dog population management · Vaccination of people at high risk of exposure to the rabies virus, e.g., laboratory staff working with the rabies virus, veterinarians and animal handlers [1,31,32] Monoclonal antibodies.

Case management Direct Observed Treatment after laboratory confirmed diagnosis
· Any age, including in pregnancy: Oral rifampicin (10 mg/kg) daily for 8 weeks and oral clarithromycin (7.5 mg/kg) twice daily for 8 weeks (including for pregnant women) · Adults only: Oral rifampicin (10 mg/kg) once daily for 8 weeks and oral moxifloxacin (400 mg) by mouth once daily for 8 weeks. Selected complementary core interventions [1] · Surgery (debridement, skin grafting, scar revision) New treatment options with reduced treatment duration and lower toxicity, especially for children.
Page 13 of 53

Gaps in anti-infective drugs as per 2030 roadmap [1]
· In case of joint involvement or movement limitation, appropriate positioning with frequent exercise. [ Case management · Treatment with albendazole or mebendazole Albendazole (drug of choice) 10-15mg/kg/day, in two divided doses, with a fat rich meal to increase its bioavailability. Mebendazole may be used at 40-50mg/kg daily, in three divided doses if albendazole is not available or not tolerated. · Other options for cystic echinococcosis include percutaneous methods + albendazole prophylaxis with the PAIR (Puncture, Aspiration, Injection, Reaspiration) technique, standard catheterization, or the modified catheterization technique, surgery (cyst removal +albendazole prophylaxis, and "watch and wait" · Other option for alveolar echinococcosis: curative surgery [1,38,39,40] Selected complementary core interventions (WHO, 2020): 'One Health' approach In collaboration with veterinary and food safety authorities · WASH · Periodic deworming of dogs with praziquantel · Livestock vaccination · where feasible, anthelminthic baiting of foxes. [1,39,40,41] Identification of optimal albendazole treatment courses (indicates that drugs with improved efficacy would add value).
Page 15 of 53

Anti-infective drugs as per 2030 Roadmap [1]
Anti-infective drug-based core strategic interventions [ Preventive Chemotherapy · Endemic populations: Single dose praziquantel (10 mg/kg) or, if active surveillance and medical referral of neurological adverse events is in place, niclosamide (2 g, dose adjusted for children), or albendazole 400 mg/day for 3 consecutive days [1,42]. · Endemic population in communities with school based preventive chemotherapy for soil-transmitted helminths and reporting system with active surveillance and medical referral of neurological adverse events: coadministration of single dose praziquantel (10 mg/kg) and single dose albendazole (400 mg) to school age children [42]. Case management · Taeniasis: Single administration of praziquantel (10 mg/kg) or niclosamide (single dose, adults and children >6 years: 2 g, children 2-6 years 1g, children < 2 years 0.5 g, after light meal followed after 2 hours by laxative) [43] Efficacy of current treatment strategies    Anti-infective drugs for diseases for which preventive chemotherapy is the main strategic core intervention strategy The strategic core intervention for many NTDs is or includes preventive chemotherapy (PC), i.e., drug administration to specified (eligible, at risk) populations without individual diagnosis. These include NTDs targeted for eradication (yaws), for elimination ( i.e., interruption of transmission, onchocerciasis, leprosy), for elimination as a public health problem (LF, schistosomiasis, STH, trachoma) and for control (food-borne trematodiases, scabies, taeniasis and cysticercosis) by or beyond 2030 (Table S2) [7]. Many drugs were originally developed for veterinary use. Prior registration for veterinary use accelerates and, to some extent, reduces costs and risk for development for human use because of significant overlap between regulatory requirements for veterinary and human drugs for non-clinical studies to characterize the drug toxicity profile (dose-response relationship, affected organs, reversibility of effects [8]). Continued large scale use of the same drug for animal and human health and development of resistance is one of the aspects that require 'One Health' approaches [7,9]. Monitoring drug susceptibility, including variability of response, is a challenge for PC programmes given the lack of suitable diagnostics for this and other purposes. The WHO NTD department has formed a Diagnostics Technical Advisory Committee. WHO makes the target product profiles and preferred product characteristics emerging from this committee as well as others available within its 'Global Observatory on Health Research and Development (https://www.who.int/observatories/global-observatory-on-health-research-and-development, https://www.who.int/observatories/global-observatory-on-health-research-anddevelopment/analyses-and-syntheses/target-product-profile/who-target-product-profiles) [7,10].

Ivermectin
Ivermectin, a macrocyclic lactone discovered in 1975 [11], is a semisynthetic anthelmintic derived from avermectin, a fermentation product of Streptomyces avermitilis. Macrocyclic lactones have activity against a broad spectrum of endo-and ecto-parasites. They are agonists of the glutamate-gated chloride channel, present in the neurons and pharyngeal muscles of nematodes and arthropods, but not of humans. Activation of the channel inhibits movement and pharyngeal pumping, leading to paralysis [12,13,14]. The role of the human immune system in the efficacy of ivermectin against filarial nematodes is still under investigation [15,16,17,18]. Consideration for development for onchocerciasis started in 1978 [19,20] before introduction into the veterinary market in 1981 [21,22]. In heavily Loa loa infected individuals, ivermectin treatment can result in severe and potentially fatal adverse reactions [23,24,25]. This prohibits ivermectin use in loiasis endemic areas that are not onchocerciasis meso-and hyperendemic due to the overall riskbenefit for the population [26]. Until the advent of drugs safe in Loa loa co-infected individuals, alternative treatment strategies are needed [27]. Concern has been raised about O. volvulus 'suboptimal response' or potentially emerging resistance to ivermectin's embryostatic effect (i.e., time to resumption of microfilariae production and release by the macrofilariae) in some regions after long term use of ivermectin [23,28,29,30,31,32,33]. However, genome-wide association analyses suggest that O. volvulus response to ivermectin is a polygenically determined quantitative trait with different identical or related molecular pathways determining the extent of ivermectin response in different O. volvulus populations [34]. Furthermore, 'suboptimal response' to ivermectin was observed in some O. volvulus infected individuals in areas without ivermectin treatment history [35,36]. This highlights the need to include variability of response in monitoring of drug response and interpretation of the results.

Albendazole, mebendazole and triclabendazole
Albendazole, mebendazole, and triclabendazole belong to the class of benzimidazoles. The class was originally developed as plant fungicides and later as veterinary anthelminthics [37]. Benzimidazole exposure results in inhibition of beta tubulin polymerase causing disruption of cytoplasmic microtubule formation [12,38]. This leads to the killing of adult stages of gut-dwelling helminths, as well as sterilization or killing of the eggs and larvae [37]. Albendazole is a broad-spectrum anthelminthic, first approved for use in humans in 1982. In its current formulation for human use it is poorly absorbed [38]. Mebendazole is also a broad-spectrum anthelminthic. Its mode of action involves multiple targets including glucose uptake in nematodes and cestodes in addition to inhibition of tubulin polymerization [39]. Triclabendazole is a narrow-spectrum anthelminthic originally developed for animal fasciolosis. Its mode of action is not completely understood. Triclabendazole and its metabolites are thought to cross the tegument of the immature and adult worms, resulting in resting membrane potential alternation, interference with microtubule structure and function, inhibition of protein synthesis and ultimately death [40,41]. Development for human fascioliasis, one of the most widespread foodborne trematode infections, was initiated in the 1990ies by WHO in collaboration with Chemische Industrie Basel (CIBA) after a fascioliasis epidemic in Iran in 1989. Regulatory approval for this indication was obtained in Egypt in 1997 and in France in 2002 [41] . Triclabendazole was approved for treatment of fascioliasis in patients 6 years or older by the US FDA in 2019 [40]. Triclabendazole is currently the only drug available able to kill early immature and adult Fasciola hepatica [41]. Triclabendazole is under consideration for repurposing for drug-resistant bacterial infections [42].

Diethylcarbamazine
Diethylcarbamazine (DEC), discovered in 1947, is a piperazine derivative anthelmintic evaluated for its efficacy and safety for onchocerciasis beginning in the 1950ies [43]. In vitro experiments at therapeutic concentrations demonstrated the loss of the microfilarial sheath with subsequent damage of organelles and apoptosis of the filarial nematode Wuchereria bancrofti [44], the cause of 90% of lymphatic filariasis cases globally [45]. The Global Programme to Eliminate Lymphatic Filariasis advocated for two elimination strategies in areas not co-endemic for either loiasis or onchocerciasis: annual mass drug administration (MDA) of a single dose of DEC or DEC with albendazole, estimated to require 4-6 years, or substitution of table/cooking salt by DEC-fortified salt (0.2-0.4% w/w) estimated to require 6-12 months [Ottesen et al. 1997]. By 2020, MDA had been implemented in at least one endemic area in 69/72 endemic countries and 17/72 countries had met the criteria for elimination of LF as a public health problem [46]. The data from studies and pilot/small scale use of DEC-fortified salt were encouraging [47,48]. However, implementation for large scale LF control may have been limited [49] to four countries: Taiwan [48], China, where elimination of LF as a public health problem has been partly attributed to the use of DECfortified salt [50], Haiti [51], and some areas in India [52,53,54].

Praziquantel
Praziquantel is a chiral pyrazine-isoquinoline derivative discovered in 1972 and first developed for veterinary use. It has a broad spectrum of activity against trematodes and cestodes [55]. The anti-helminthic action is not fully understood and may include binding to calcium channels, tegument disruption, binding and polymerization of actin and exposure of surface membrane antigens [56,57,58]. As for other NTDs, the extent to which long-term preventive chemotherapy affects parasite drug susceptibility is unknown [59]. The WHO recommended single dose of 40 mg/kg praziquantel (Table S2) achieves 95%, 94.1% and 86.3% ERR in Schistosoma. japonicum, S. haematobium and S. mansoni, respectively [57]. A dose of 60mg/kg did not increase efficacy against S. mansoni or S. japonicum [60,61,62]. Known limitations of praziquantel are its inactivity against immature parasites [57].
Praziquantel is well tolerated, but in individuals with cysticercosis and cysts in the central nervous system or eyes, the inflammatory reaction to dying Taenia solium can result in seizures, and/or cerebral infarction and permanent eye lesion [57]. The commercially available tablets include both (R)-praziquantel (L) with anthelmintic activity and the inactive (S)-praziquantel (D) which contributes to the bitter taste and a 600 mg tablet size that is unsuitable for pre-school children [63]. The Paediatric Praziquantel Consortium (https://www.pediatricpraziquantelconsortium.org/) has developed a paediatric formulation [64,65].

Azithromycin
Azithromycin is a macrolide antibiotic with a 15-member lactone ring structure with two sugars attached via a glycosidic bond that is semi-synthetically produced from erythromycin A. It is the single compound in its azalide subclass. Azithromycin binds to the 50S ribosomal subunit at the peptidyl transferase centre, preventing protein synthesis [66,67]. Besides broad-spectrum activity against Gram-positive and Gram-negative bacteria, azithromycin has activity against the apicomplexan parasites Toxoplasma gondii and Malaria spp. [67,68,69,70]. Azithromycin can be taken orally and has a safety profile [71,72] which supports inclusion of pregnant women and children in MDA [7]. The absence of drug-drug interactions, allows integrating azithromycin, ivermectin, diethylcarbamazine and albendazole MDA [71]. Since studies in Papua New Guinea and Ghana [73,74] showed non-inferiority to penicillin G, azithromycin is the preferred antibiotic for treating yaws [74,75,76]. A single 20 mg/kg dose is highly effective in treating trachoma infections [77,78]. Yaws and trachoma can be co-endemic and research has shown that 20 mg/kg is as effective as 30 mg/kg against Yaws [79].

Benzathine penicillin
Benzathine penicillin (penicillin G) was discovered in 1951 [80]. It is a bactericidal betalactam that inhibits bacterial peptidoglycan transpeptidases, preventing cell wall formation during cell division [81]. While oral formulations are available, benzathine penicillin is frequently administered intravenously or intramuscularly due to its poor oral bioavailability. Slow-release formulations provide effective serum levels measurable for at least 14 days. Benzathine penicillin has a broad spectrum of activity against Gram-positive and Gramnegative bacteria, including the causative agents of yaws and trachoma [75,80]. Despite the need for trained health professionals and discomfort of injections for the patient, benzathine penicillin mass treatment of cases and contacts was implemented in 46 countries from 1952-1964 and reduced global yaws and other treponematoses burden by 95% [75]. Care must be taken to ensure individuals with known penicillin sensitivity or history of allergic reactions, including anaphylaxis, are excluded and immediate access to required interventions is available to avoid fatalities [64,65].

Permethrin
Permethrin is a synthetic pyrethroid insecticide, based on pyrethrum extracts, designed to increase insecticidal activity, lower mammalian toxicity and provide the photostability required for agricultural use. Permethrin acts on the nerve cell membrane of arthropods to disrupt the sodium channel current that regulates the polarization of the membrane. This results in delayed repolarization and subsequent paralysis and death of the parasites. Permethrin is an active ingredient of mosquito nets [82,83,84,85,86].
Permethrin is available in topical products for human use. The WHO EML 2021 lists a 1% lotion and a 5% cream [1]. A permethrin cream (5%) was approved for the treatment of scabies in children two months of age or older by the US FDA in 1989 [87]. Safety in younger infants is an open question [88,89] The absorption of permethrin through the skin is limited to 2% of the amount applied with the fraction absorbed being eliminated via rapid metabolism [87,88]. Five percent (5%) permethrin cream applied head-to-toe including in intimate areas is highly effective for treatment of scabies cases and reducing the risk of infection of contacts [90]. However, large scale use in endemic community settings faces a number challenges ranging from cost to individual acceptance and compliance with the treatment regimen [91,92,93,94]. This limits its utility and drives considerations for use only in individuals for which ivermectin is contra-indicated [91,95,96].

Benzyl benzoate
Benzyl benzoate is an ester of benzoic acid and benzyl alcohol which is neurotoxic to mites [97]. In vitro, 100% of mites were killed after 3 hours of exposure to 25% benzyl benzoate [98]. The drug has been used for scabies since the late 1930ies [99] and is available as 10-25% lotions or emulsions [100,101]. Characterized as safe in children ≥1 month [102], a 5% topical cream was identified as the treatment of choice for infants >2 months for whom safe and effective ivermectin doses have not been identified and as an option for infants younger than 2 months with application for four hours [96]. Severe skin irritation can occur within minutes of application [101]. An informal consultation on a framework for scabies control was held by WHO in 2019 to review current data and gather expert views. The experts recommended that, in view of the inferior efficacy and higher rate of adverse effects of benzyl benzoate compared to permethrin, benzyl benzoate should only be used when topical treatment is indicated (i.e. for individuals for which ivermectin is not approved) and permethrin is unavailable [91,95,96].

Malathion
Malathion is an organophosphate insecticide. Its toxic metabolite malaoxon irreversibly inhibits acetylcholinesterase, resulting in acetylcholine accumulation and disruption of the nervous system function [103,104,105]. Malathion, as an aqueous lotion 0.5% w/v, was first developed for human use in the treatment of headlice. The justification for the use of malathion in scabies comes from a study conducted in 1978 that demonstrated an 83% cure rate in a population of 30 individuals with scabies [106]. A 2013 review did not identify sufficient evidence to assess the relative efficacy of malathion and other scabies treatment options [107]. Malathion is listed as an alternative rather than a recommended treatment of scabies in the European recommendations [108]. In 2015 malathion was classified as probably carcinogenic to humans by the WHO International Agency for Research on Cancer [105,109].

Sulphur ointment
Sulphur is the oldest antiscabietic, reported to have been used already around 25 AD. The reduction of sulphur to hydrogen sulphide by bacteria on the skin results in killing the scabies mite [97]. Topical sulphur treatments for scabies contain between 2-33% sulphur. They were used in the youngest children (at strengths of <10% sulphur), including those under 2 months as well as pregnant women due to a perceived safety profile resulting from the lack of absorption following dermal application [97]. It was recently used during MDA for a scabies outbreak in Ethiopia for treatment of children under 10 years of age and pregnant and breastfeeding women [110].
The efficacy of sulphur is inferior to that of permethrin and ivermectin [97]. Sulphur is listed as an alternative rather than a recommended treatment of scabies in the European recommendations [108]. Its use can be limited by its strong and unpleasant odour that results from the formation of hydrogen sulphide as well as the fact that it can stain clothing [111].

Anti-infective drugs for diseases for which case management is the main
control-and elimination strategy 2.1 Antibiotics

Rifampicin
Rifampicin, derived from Nocardia mediterranei, was discovered in 1957 and synthesized in 1965. Activity against Mycobacterium tuberculosis was determined in vitro and in vivo in mice, guinea pigs, and rabbits before trials in humans, initiated in 1966, demonstrated efficacy against M. tuberculosis strains resistant against all other anti-tuberculosis drugs at the time [112]. Rifampicin inhibits bacterial DNA-dependent RNA synthesis, thus preventing transcription and inhibiting bacterial protein synthesis, resulting in a bactericidal effect. Differences in antimicrobial activity against Gram-positive and Gram-negative bacteria are not related to different binding sites on the RNA polymerase but to other factors like efflux pumps. Different mechanisms of resistance have been described, including changes of the binding pocket of rifampicin on the RNA polymerases [113]. Rifampicin activates the nuclear pregnane X receptor increasing the expression of genes whose products are involved in drug metabolism and transport, inducing cytochrome P450 2B6 (CYP2B6), CYP 3A4 and P-glycoprotein. The resulting potential for drug-drug interaction needs to be considered in particular in treatment of TB and HIV co-infected patients [114]. A systematic review concluded that a single rifampicin dose reduced leprosy incidence in contacts of patients in the first two years by 57% [115]. Rifampicin-associated adverse effects include flu-like syndrome, gastrointestinal and dermatological events, as well as hepatitis and cholestasis [112].

Dapsone
Dapsone, discovered in 1908, is a sulphone bacteriostatic antibiotic and anti-inflammatory agent. Its bacteriostatic effect is due to its sulphonamide-like ability to compete with paraaminobenzoic acid for the active site of dihydropteroate synthetase, thus inhibiting dihydrofolic acid synthesis. The anti-inflammatory effect may be based on different mechanisms including inhibition of chemokine production, neutrophil response to chemotactic signals and adherence to endothelium, generation of toxic and oxygen-derived radicals. Dapsone is metabolized by cytochrome P450, making it susceptible to drug-drug interactions. The resulting hydroxylamines are considered responsible for the dose-dependent adverse effects agranulocytosis, methemoglobinemia and haemolysis. The most important other dose-dependent adverse effects occurring at low frequency within the dose range considered effective for leprosy is peripheral neuropathy. Life-threatening dapsone hypersensitivity syndrome occurs in 0.5% to 3.6% of patients [116]. This syndrome has been linked to HLA-B*13-1 polymorphism which could provide a path to pre-treatment screening [117,118,119]. Dapsone resistance of Mycobacterium leprae has been known since 1977 [120,121] and has been attributed to a mutation of folP1, a gene coding for dihydropteroate synthase [122].

Clofazimine
Clofazimine, first described in 1957, is a riminophenazine antibiotic. It is active against slowly and rapidly growing mycobacteria, as well as many other Gram-positive bacteria in vitro but not against Gram-negative bacteria. The primary site of action has been proposed to be the outer membrane. Putative targets include ion transporters and the bacterial respiratory chain. Besides anti-infective activity, clofazimine also has anti-inflammatory properties benefitting the treatment of leprosy. Its high lipophilicity enables clofazimine to accumulate in skin and nerves which contributes to its efficacy against erythema nodosum leprosum. Adverse effects of clofazimine include reversible discolouration of the skin and conjunctiva and gastrointestinal events which are usually mild to moderate but may in some cases be severe including bleeding, splenic infarction, and bowel obstruction [123,124]. The primary mechanism of resistance was described to be a mutation in the rv0678 gene, a gene that encodes a transcriptional repressor for the efflux pump MmpL5 [125].

Clarithromycin
Clarithromycin is a macrolide antibiotic which differs from erythromycin through substitution of the hydroxy group at the lactone ring by an O-methyl group. This confers greater acid stability resulting in better oral availability and may contribute to improved intracellular activity. Clarithromycin is metabolized by hepatic cytochrome P450 enzymes. Its metabolite 14-hydroxy-clarithromycin has activity with additive or synergistic activity with clarithromycin. Macrolides inhibit protein synthesis by reversible binding to the 50S ribosomal subunit of susceptible bacteria [126] and have immunomodulatory effects [127].
The potential for drug-drug interactions is based on binding and inhibition of macrolides to cytochrome CYP3A4 isoforms. Clarithromycin has less affinity to CYP3A4 than erythromycin [128,129]

Moxifloxacin
Moxifloxacin is a fluoroquinolone antibiotic, approved for human use by the US FDA in 1999 (https://www.accessdata.fda.gov/drugsatfda_docs/nda/99/21-085_Avelox.cfm). Compared to fluoroquinolones available at that time it has enhanced activity against Grampositive and atypical bacteria with a comparable spectrum of activity against Gram-negative bacteria. Fluoroquinolones inhibit DNA replication by affecting DNA gyrase and topoisomerase IV with topoisomerase IV being the primary target in Gram-positive bacteria and DNA gyrase that in Gram-negative bacteria. Data obtained in S. pneumoniae and E. coli suggest moxifloxacin may have equal and simultaneous activity on both enzymes. Fluoroquinolone resistance is based on alteration of the genes coding for the target enzymes and the gene coding for the efflux pump [130].

Tetracycline
Tetracycline is an antibiotic that was discovered in 1953. The term has also been used to describe the structurally related family of antibiotics, which inhibit the bacterial protein biosynthesis by preventing the attachment of the aminoacyl-tRNA to the ribosomal acceptor site. They are broad-spectrum antibiotics, exhibiting activity against a wide range of Grampositive and Gram-negative bacteria, as well as atypical pathogens like Chlamydia [131]. Before the mid-1950s, pathogens resistant to tetracyclines were rare, but since then, many different tetracycline-resistant genes have been characterized. [131].

Fexinidazole
Fexinidazole is 2-substituted 5-nitroimidazole synthesized in the 1970s by Hoechst AG (now Sanofi). Its anti-trypanosomal activity was initially identified in the 1980s and later confirmed [132,133]. Fexinidazole is metabolized rapidly to sulfoxide and sulfone derivatives [132,134]. The subsequent metabolism and mechanism of action in trypanosomes are unknown [135].  [135,136,137]. In 2019 fexinidazole was added to the WHO EML and included in the 'WHO interim guidelines for the treatment of gambiense human African trypanosomiasis' [138]. In 2020, fexinidazole received US FDA approval [139]. In contrast to all other available treatments for T.

Pentamidine
Pentamidine, a synthetic aromatic diamidine, was introduced in 1940 [140]. The exact target and mode of action is unknown, but may include DNA binding/damage, loss of kinetoplast DNA, and disruption of mitochondrial membrane potential [141,142]. Loss of kinetoplast DNA has been suggested to precede the loss of mitochondrial membrane potential, but it is unclear if this effect is strictly sequential or if pentamidine also has direct effects on the mitochondrial membrane [141]. Despite 80 years of use, it is still highly effective (cure rate = 93-98%) in treating first-stage T. b. gambiense HAT [140,143], but today is the 1 st line treatment only for children <6 years or <20 kg for whom fexinidazole is not yet registered. Pentamidine is administered once daily intramuscularly for 7-10 days [140]. If given intravenously, care must be taken that it is not given as a bolus, but rather slowly over 60 minutes to avoid a possible induction of hypoglycemia. An oral analogue of pentamidine had comparable efficacy to the injected form, but was too toxic [144]. As there is little economic incentive in pentamidine, its production was almost discontinued, but in 2001, Sanofi-Aventis agreed to continue producing it for the WHO [143]. It cannot pass the blood-brain barrier and therefore is not effective for second-stage infections [140].

Suramin
Suramin is a polysulfonated napththyl urea introduced to treat T. b. gambiense and T. brucei rhodesiense HAT in 1922 and is one of the first anti-infective agents developed from trypan blue and trypane red in one of the first medicinal chemistry programs at Bayer [140,145]. Due to its six negative charges at physiological pH, it does not pass the blood brain barrier and, therefore, was only effective against first-stage trypanosomiasis disease [145]. Suramin is a multifunctional compound with activity against other parasitic diseases, viruses, cancers, and snakebites, and even autism [145]. Due to the many targets, the mode of action of suramin is not well understood in general and even less-so in trypanosomes [145]. In trypanosomes it has been shown to inhibit cytokinesis as demonstrated by cells with two nuclei [141]. Suramin has been shown to inhibit glycolytic enzymes and inhibit oxidative phosphorylation, although it is not understood how the large and highly negatively charged molecule can pass the membranes of the glycosomes and mitochondria where the enzymes are located [145]. Life threatening reactions to the 7-day course for T. b. rhodesiense HAT and lethal outcomes are rare. Pyrexia and usually mild and reversible nephrotoxicity are driven by concentrations in the kidneys [140]. As T. b. rhodesiense HAT progresses quickly to the second stage, treatment of pregnant women with first-stage HAT cannot be delayed until after birth of the child [140]. Suramin administered for 6 weeks is macrofilaricidal in O. volvulus, but the adverse reactions make it unsuitable for large scale use [146,147]. Recent research shows activity against Leishmania major and L. donovani, and that suramin can block host cell invasion by Plasmodium falciparum [145]. Suramin is instable in air and must be administered for HAT by slow intravenous injection every 3-7 days for 4 weeks [140,144].

Melarsoprol
Melarsoprol is a trivalent organic arsenical compound that, since 1949, was the first-line drug of choice over other arsenic derivatives to treat second-stage T.b. gambiense and T. b. rhodesiense HAT [140,143,144]. The mode of action of melarsoprol is still being elucidated. It forms adducts with trypanothione and is an indiscriminate inhibitor of kinases (mainly dithiol containing trypanosomal enzymes); the latter indicating involvement of signaling cascades [140,141]. Treatment of trypanosomes results in a defect in mitosis shown by an increased number of cells with replicated but unsegregated nuclear genomes [141]. The compound is liposoluble and administered intravenously in propylene glycol, an irritant. This makes melarsoprol not only difficult to administer, requiring hospitalization, but painful to the patients [143]. For several decades, melarsoprol treatment followed the regimens of other arsenicals and varied from country to country, including serial drug application with 1-week intervals without drug [140,148]. Pharmacokinetic studies supported the hypothesis that a shorter, uninterrupted treatment regimen could be equally effective [149,150]. This informed studies which showed the safety and efficacy of a 10-day treatment for second stage T. b. gambiense HAT [149,150,151] and T. b. rhodesiense HAT [148]. The most feared adverse reaction is reactive encephalopathy that can occur in up to 10% of patients with a median fatality rate of 50% [140,143]. Melarsoprol is still the only drug available to treat second-stage T. b. rhodesiense HAT [140] but is today only a rescue treatment for T. b. gambiense HAT patients who have failed treatment with Nifurtimox-Eflornithine Combination Treatment (NECT) and fexinidazole [138]. There are few data on the safety of melarsoprol during pregnancy, but theoretically it is contraindicated. However, due to the severity of T. b. rhodesiense HAT, its use as treatment cannot be delayed until after the birth of the child [140].

Eflornithine
Eflornithine (D,L-α-difluoromethly ornithine) was at one time evaluated as a cancer drug. It's antitrypanosomal effect was discovered in 1980 through WHO/TDR funded studies on the polyamine metabolism of trypanosomes [152]. Eflornithine irreversibly inhibits ornithine decarboxylase of trypanosomes [143], blocking production of the polyamine putrescine and subsequent DNA synthesis. This results in the parasites entering a dormant state susceptible to the host immune system [140]. Trypanosomes are more sensitive to the drug than mammalian cells, probably due to a slower ornithine decarboxylase turnover rate [144]. Eflornithine's effect on patients with second stage T. b. gambiense HAT [153] earned it the name 'resurrection drug'. Despite that, and the fact that public funding provided by WHO/TDR contributed to eflornithine development [153,154], eflornithine was no longer available after the manufacturer Marion Merrell Dow merged with Hoechst and Roussel. WHO was not able to identify an alternative manufacturer for an affordable price [152,155].
It took the discovery that eflornithine was being manufactured for a cream to remove unwanted facial hair in women, which had received regulatory approval in 2000 [156], and subsequent intense advocacy by Médecins Sans Frontières and WHO for eflornithine to become available again to save the lives of patients with HAT [152,157,158]. Currently, supply until 2025 has been secured through renewal of the WHO and Sanofi agreement [159]. Eflornithine is not used to treat second-stage T. b. rhodesiense HAT as these parasites are less susceptible to the compound [143]. Although an oral formulation was available, it did not ensure high enough levels in the cerebral spinal fluid, and eflornithine is therefore given intravenously [140]. As monotherapy, it requires administration 4 times a day every six hours for 14 days [144], which is difficult in the health centers in the remote rural areas where HAT is endemic. Through combination with 10 days of oral nifurtimox treatment every 8 hours (NECT), eflornithine eflornithine infusion can be reduced to twice daily for 7-days [160,161]. Little information is available on the safety of eflornithine during pregnancy and, when possible, watchful waiting (regular monthly clinical assessment) is used. If the health of the mother is at risk, eflornithine or NECT is given, otherwise pentamidine is used after the first trimester to prevent infection of the fetus [138,140].

Benznidazole
Benznidazole is a nitroimidazole antiparasitic drug for treatment of acute and early chronic Chagas disease [7]. It is reduced to reactive metabolites by nitroreductases, but the mechanism of its action is not well understood [162,163,164]. Despite availability since 1971, its role in the treatment of chronic disease remains under discussion [165,166]. Its better tolerance in infants and children than adults has been attributed to a faster hepatic elimination compared to adults [167]. A paediatric formulation was developed and registered in Brazil in 2011, in the US in 2017 (for ages 2-12 years https://www.accessdata.fda.gov/drugsatfda_docs/nda/2017/209570Orig1s000TOC.cfm, accessed 20 June 2022) , and in Argentina in 2018 [168]. A recent study concluded that shorter treatment durations and/or lower doses of benznidazole could have similar antiparasitic effects with better tolerability than the current standard treatment, a promising finding to be confirmed via further studies [169,170].

Nifurtimox
Nifurtimox is a 5-nitrofuran derivative, introduced for treatment of Chagas disease in the late 1960ies [171]. Following demonstration of the efficacy and safety of NECT, the combination of oral nifurtimox with intravenous eflornithine, for treatment of second stage T. b. gambiense HAT [160,161], this combination was added to the WHO EML in 2009 [172] and became the treatment of choice for this indication [158,173]. Nifurtimox was added to the WHO EML for children for African trypanosomiasis in 2013 [174] after data became available demonstrating that in children NECT efficacy is comparable and safety comparable or better in children than adults [173,175,176,177]. The mechanism of action for both T. brucei and T. cruzi involves reduction by an NADH-dependent bacterial-like nitroreductase and generation of a cytotoxic, unsaturated open-chain nitrile derivative [133].

Amphotericin B and liposomal amphotericin B
Amphotericin B is a polyene antibiotic with efficacy against fungal infections as well as T. cruzi, Schistosoma mansoni, Echinococcus multilocularis and Leishmania spp [184]. The long-established notion that Amphotericin B mechanism of action is via cell membrane pore formation after binding to ergosterol has been questioned in favour of pleiotropic effects including induction of oxidative damage and an immunomodulatory effect [184]. It is available in several formulations: deoxycholate solution, colloidal dispersion with cholesterol sulphate, a lipid complex with two phospholipids, and in unilamellar liposomes formed from cholesterol and other phospholipids [178,184,185]. Liposomal amphotericin B was originally developed for severe systemic and deep mycoses. It was first used to treat a European with mediterranean visceral leishmaniasis who had failed treatment with antimonials, pentamidine and paromomycin [186,187]. This and subsequent experience motivated a clinical development programme to obtain the data needed for registration of liposomal amphotericin B for treatment of visceral leishmaniasis in collaboration between the company and WHO/TDR [186]. Liposomal amphotericin B (Ambisome) was approved by the US FDA in 1997 for three indications including visceral leishmaniasis (https://www.accessdata.fda.gov/drugsatfda_docs/nda/97/050740_ambisome_toc.cfm). A single dose of liposomal amphotericin B has been shown to be efficacious as well as safe and effective for visceral leishmaniasis in India and Bangladesh [188,189,190] and has become the preferred first-line treatment, replacing miltefosine [191]. Further studies are needed to define the optimum treatment liposomal amphotericin B regimen for other clinical presentations, pathology, causative species, geographic region, use history and co-infections. [192].

Miltefosine
Miltefosine (hexadecylphosphocholine) is an alkyl phosphocholine compound and a structural analogue of lecithin [193]. It was evaluated from the 1980ies as a cancer drug resulting in registration of a topical formulation for treatment of skin lesion from breast cancer [194,195]. Research into miltefosine's antileishmanial effect also dates back to the 1980ies [196]. Mechanisms involved in its antileishmanial action include incorporation into membrane lipid bilayers, disturbance of membrane metabolism and induction of apoptosis-like cell death as well as host-mediated immunomodulation [193]. Dose limiting gastrointestinal adverse reactions during long treatment resulted in discontinuation of development of oral formulations for cancer. The pre-clinical as well as clinical data obtained during that development [194] provided a valuable basis for development of miltefosine as the first oral treatment for visceral leishmaniasis [197]. Miltefosine is teratogenic in rats, but not rabbits. Contraindication during pregnancy is one of its main limitations [194,198]. The availability of an oral treatment was one of the factors that lead to a regional strategic framework for elimination of visceral leishmaniasis from the Indian Subcontinent [199,200]. Miltefosine development for treatment of visceral leishmaniasis was accomplished in collaboration between Asta Medica (Zentaris, Germany) and WHO/TDR with WHO/TDR providing both relevant expertise as well as funding. The collaboration was based on a Memorandum of Understanding concluded in 1995 between WHO and the company which included provisions for availability and affordability of miltefosine for the public health systems in endemic countries, should development be successful and result in registration [195,201]. Registration was achieved initially in Germany and in India in 2002 and later in other endemic countries. However, affordable access was only temporary. In contrast, the incentives provided by the US congress for investment into the development of drugs for neglected diseases via the 'Priority Review Voucher' (https://www.fda.gov/about-fda/centerdrug-evaluation-and-research-cder/tropical-disease-priority-review-voucher-program) resulted in a company (Knight Therapeutics) who had never invested in miltefosine development, nor committed to making miltefosine available at affordable prices, benefitting from these incentives to the tune of US$125 Million [195,202]. This has resulted in calls to change the conditions under which a 'Priority Review Voucher' is awarded [202,203].

Paromomycin
Paromomycin, a highly hydrophilic and lipid insoluble aminoglycoside also known as aminosidine, is a broad-spectrum antibiotic . Its antileishmanial effect was discovered in 1960 [180]. The mechanism of action of paromomycin is inhibition of protozoan protein synthesis by binding to the 30S ribosomal subunit resulting in the accumulation of abnormal 30S-50S ribosomal complexes and finally causing cell death [180]. Paromomycin can be used in pregnant leishmaniasis patients [180].

Itraconazole
Itraconazole is a broad-spectrum antifungal azole drug, patented in 1978 and approved by the US FDA in 1992 (https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=overview.process&ApplN o=020083, accessed 28 June 2022). Azoles inhibit the synthesis of ergosterol via the inhibition of lanosterol 14α-demethylase resulting in fungal membrane destruction. Itraconazole also impacts several metabolic pathways important for human cell proliferation resulting in interest in repurposing itraconazole for cancer therapy [211] Itraconazole, like other azoles, is metabolized by cytochome P450 3A4. Therefore, drug-drug interactions with other drugs metabolized by CYP450 3A4 need to be considered [212].

1.
World Health Organization. The selection and use of essential medicines: Report of the WHO Expert Committee on Selection and Use of Essential Medicines, 2021 (including the 22nd WHO model list of essential medicines and the 8th WHO model